Alternative splicing is one of the major mechanisms by which genes generate multiple protein isoforms with potentially different activities. Such activity differences may depend directly on the presence or absence of a distinct protein domain or on post-translational modifications associated with the alternatively included domains. The gene Mitf, encoding a basic helix-loop-helix-leucine zipper transcription factor with critical roles in the biology of melanin-bearing pigment cells, is an excellent example showing the intricate interplay between alternative splicing and post-translational modifications. The gene's exon 2B contains the codon for serine-73 whose MAPK-dependent phosphorylation has previously been implicated in the regulation of MITF protein activity and stability based on in vitro experiments. In wild-type mice, exon 2B is present in over 90% of Mitf RNA but in mice carrying a targeted serine-73-to-alanine mutation, it is present in only 10% of such RNA. The difference is due to the action of the serine/arginine-rich protein SRSF5 which binds the sequence containing the wild-type codon with higher affinity than that containing the mutated codon. To dissociate serine-73 phosphorylation from splicing regulation, we generated additional targeted mutants in which splicing-out of exon 2B is abolished by a translationally silent alteration of the 5 splice site and which carry either a wild-type serine codon or one encoding an alanine or a phosphomimetic aspartate. While in each of the corresponding mouse lines Mitf RNA is expressed at comparable levels, MITF protein accumulates to slightly different levels as anticipated from the distinct protein stabilities associated with the different mutations. However, phenotypic differences in coat color can be revealed only when RNA levels are slightly reduced by the presence of a neo-cassette or when the targeted mutations are combined with other mutant Mitf alleles. For instance, homozygotes carrying the neo-cassette have large white belly spots when their forcibly expressed exon 2B encodes a wild-type serine but none when it encodes an alanine. In certain allelic combinations, the alanine mutation can even lead to a darker pigmentation compared with a corresponding combination with a wild-type Mitf gene. The results can be explained by a combination of the effects of Ser-73 phosphorylation-dependent protein levels and the presence or absence of exon 2B. They show that alternative splicing of exon 2B of Mitf is a critical determinant of melanocyte physiology and suggest that in populations segregating allelic variants of a gene, such as in humans, a mutant copy of a gene may produce different phenotypes depending on what variant copy it is combined with.